Sulfuryl-transfer, much like phosphoryl-transfer, is an important and prevalent regulatory mechanism in mammalian metabolism; yet, little is known about the enzymology of sulfuryl-transfer. Estrogen sulfotransferase (EST) catalyzes transfer of the sulfuryl-moiety from PAPS, or activated sulfate, to a variety of estrogens (1). PAPS2- + E PAP2- + ES- + H+ (1) Sulfating estrogen regulates its cellular activity by preventing it from binding to and activating the estrogen receptor. EST expression regulates the normal estrogen response of the endometrium and breast during the menstrual cycle. Recent studies suggest a tight, casual link between EST expression and the estrogen-dependent growth response that underlies tumorgenesis in both breast and endometrium. The sulfotransferase are a very highly conserved enzyme family, and examples of the importance of sulfuryl-regulation and abundant. The primary goal of this proposal is to create a state-of-the-art understanding of the chemistry and enzymology of sulfuyl- transfer. This new and fundamental information will have a significant impact on the sulfotransferase field. The enzymology will be brought to bear on a topic of considerable public concern: estrogen replacement therapy. Premarin, the most widely prescribed estrogen replacement therapeutic in the United States, is injested by 10 million women daily. The specificity of human EST toward the equine estrogens found in Premarin will be determined to assess whether they are regulated via sulfation at therapeutic concentrations.